Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis, maintaining sperm production throughout adult life. The molecular mechanisms that control the critical self-renewal vs. differentiation fate decision of SSCs are largely unknown. The hypothesis is that transcription factors primarily restricted to stem and progenitor spermatogonia form regulatory networks to execute gene expression programs important for SSC fate decisions and spermatogenesis. Three Specific Aims will test this hypothesis. Experiments in Specific Aim 1 will employ functional genetics to determine if specific transcription factors are essential for spermatogenesis using a knockdown strategy in ex vivo SSC cultures followed by transplantation to assess stem cell and spermatogenic activity. Specific Aim 2 will identify the target gene repertoire of SSC transcription factors by ChIP-Seq and reveal conserved and unique targets sets. Experiments in Specific Aim 3 will confirm mechanisms of target gene regulation by EMSA and transient transfection transcriptional analysis. These results will form the basis of gene regulatory network models incorporating complex interactions from multiple factors contributing to gene regulation and SSC potential. The proposed studies will be enhance the independent career trajectory of the PI (Brian Hermann) by establishing a new research focus investigating the fundamental regulation of SSCs in the rodent testis under the mentorship of Dr. Kyle Orwig. This is a new research direction for the PI who has established expertise in Sertoli cell gene regulation and primate spermatogenesis and stem cells. The intellectual and technical environment for research in reproductive biology and stem cells at the Magee-Womens Research Institute and University of Pittsburgh is outstanding, all resources required to complete the studies are available, and there is a strong reputation for promoting development of new investigators. This combination of a PI with outstanding potential for independence, an innovative mentor, and superior environment will facilitate all aspects of the proposed studies and advancement of the PI's career towards independence. The long-term career goals of the PI are to establish an independent research program in a tenure-track faculty position, make significant scholarly contributions to the understanding of SSCs and spermatogenesis, and develop new approaches for treating male infertility. Three immediate objectives to promote the transition to independence are addressed by the career development award: 1) develop an independent research niche, 2) demonstrate outstanding productivity, and 3) secure additional independent funding. The career development plan involves conceptual mentoring in functional genetics, technical training relating to culture and transplantation of SSCs in the mouse model, instruction in bioinformatics data analysis, informal and structured mentoring in areas relevant to running a successful independent research program, and evaluation of career development progress by an external advisory board. Ultimately, these studies may provide insights about the mechanisms that initiate and maintain spermatogenesis, which has implications for treating male infertility. Investigating the biological properties of SSCs may also expand the understanding of how stem cells behave and contribute to the normal function of a variety of adult tissues. Moreover, additional nurturing of the candidate's career development will promote a competitive, independent research career to contribute substantively to the biomedical sciences. PUBLIC HEALTH RELEVANCE: Spermatogonial stem cells (SSCs) are responsible for sperm production throughout adult life and are essential for male fertility. The methods which instruct these cells to behave like stem cells and maintain sperm production are unknown. The unique combination of genes expressed by SSCs and the proteins that turn these genes on and off may control of SSC behavior. This application will provide key insights about the role of gene regulation in normal SSC behavior, which may reveal underlying causes of male infertility.